Summary: Recent studies from this laboratory using the neuronal cell line H32, which expresses endogenous V1 VP receptors, showed that activation of endogenous V1 VP receptors prevents serum deprivation-induced apoptosis, through phosphorylation-inactivation of the pro-apoptotic protein, Bad, and consequent decreases in cytosolic cytochome c and caspase-3 activation. These actions of VP are largely mediated by mitogen activated protein (MAP) kinase and extracellular signal-regulated kinases (ERK), but also involves other signaling pathways. Studies during the past year have examined the role of protein kinases C (PKC) and B (PKB) mediating VP induced antiapoptosis in H32 cells. Serum deprivation increased PKC&#948;but not PKC&#61537;or PKC&#61538;activity, while VP increased PKC&#61537;and PKC&#61538;without affecting PKC&#948;activity. Inhibition of PKC&#948;prevented caspase-3 activation, indicating that PKC&#948;mediates the proapoptotic actions of serum deprivation. Simultaneous inhibition of PKC &#61537;&&#61538;and MAP kinase abolished VP-induced Bad phosphorylation, but it only partially prevented caspase-3 inhibition. Complete abolition of the protective effect of VP on serum deprivation-induced caspase 3 activity required additional blockade of PI3K/protein kinase B (Akt). The data demonstrate that VP exerts antiapoptosis through multiple pathways;while PKC&#61537;&&#61538;together with ERK/MAP kinase activation mediates Bad phosphorylation (inactivation), the full protective action of VP requires additional activation of PKB (PI3K/Akt) pathway. The studies in H32 cells demonstrate that VP has antiapoptotic actions in a neuronal cell line and suggest that VP has neuroprotective actions. This hypothesis was tested in primary cultures of hippocampal neurons by examining the ability of VP to prevent nutrient deprivation- or glutamate induced apoptosis, and the signaling pathways involved in this effect. Deprivation of trophic factors (by changing the defined media supplement by 0.1%BSA) for 24 hours or exposure to glutamate (3 to 10 &#61549;M) caused significant decrease in neuronal cell viability and an increase in caspase-3 activity, consistent with apoptotic cell death. The effect of trophic factors deprivation or glutamate was significantly reduced by addition of 10nM VP, suggesting that VP exerts anti-apoptotic effects in neurons. This was confirmed by the ability of VP to prevent trophic factor deprivation-induced Tdt-mediated dUTP nick-end labeling (TUNEL) staining neurons. The protective effect of VP was completely blocked by V1 receptor antagonist, (Phenylac1,D-Tyr(Et)2,Lys6,Arg8,des-Gly9)-Vasopressin, indicating that it is mediated via V1 VP receptors. The anti-apoptotic effect of VP in neurons involves mitogen activated protein (MAP) kinase and extracellular signal-regulated kinase (ERK) and the PI3 kinase/Akt signaling pathways. Co-incubation of nutrient deprived neurons with VP and the selective MAPK inhibitor, U0126, or the PI3 kinase inhibitor, LY294002, reduced the inhibitory action of VP on caspase-3 activity and TUNEL staining neurons. Western blot analysis revealed biphasic increases in phosphorylation/activation of ERK during incubation with VP, increasing at from 2.5 to 5 min returning to basal by 10 min and increasing again from 30 min to 1 h. This data shows that VP has anti-apoptotic actions in neurons, an effect which is partially mediated by the MAPK signaling pathway. The study supports the view that VP plays a role as a neuroprotective agent in the brain.